A PLASMA FACTOR FROM A PATIENT WITH A BLEEDING TENDENCY CAUSES PLATELET SECRETION IN THE ABSENCE OF EXTRACELLULAR CALCIUM
A woman with a history of bruising and bleeding but with a normal platelet count and normal clotting factors, had platelets that appeared grey when stained and viewed under the microscope. Unlike the grey-platelet syndrome, the abnormality was only evident when the blood had been collected into EDTA andnot when citrate or heparin was used as anticoagulant. When we examined the EDTA-blood further we found that large quantities of beta-thromboglobulin andserotonin (5HT) were present in the plasma with only small quantities in the platelets. Hie reverse was the case for blood collected into citrate or heparin. LDH (a cytoplasmic marker) levels in EDTA-plasma were not raised.Platelet-rich plasma (PRP) was prepared from blood collected into heparin and labelled with ^-4C-5HT. Incubating the PRP with EDTA (4mM) or EGTA (3mM, sufficient to chelate all the plasma calcium but not the magnesium) caused extensive release of 14C-5HT from the platelets. When Ca++ was removed by passing the PRP through an ion-exchange resin, extensive 14C-5HT release also occurred. Hie release reaction induced by exposing the platelets to a low-Ca++ environment could be prevented by agents that increase cAMP levels.In a series of cross-over experiments, we discovered that the platelet secretion that occurred on removing Ca++ was caused by a plasma factor. Platelets from a healthy donor which had been labelled with 14C-5HT were resuspended in the patient's heparinised plasma. Incubating the platelet suspension with EGTA resulted in extensive release of 14C-5HT. Heparinised plasma from the patient was also passed through a column of Protein A-Sepharose to remove the immunoglobulin fraction. EGTA-challengof control platelets resuspended in the eluted plasma did not cause any release of 14C-5HT, suggesting that the plasma factor responsible may be an immunoglobulin.We have yet to discover whether this new abnormality (that on initial investigation could be confused with the grey-platelet syndrome) has any relevance to the in vivo bleeding situation in the patient in whom it was discovered.